A fluorescence-based imaging technique for monitoring single molecules could lead to a better understanding of how molecules are assembled, function, and interact, aiding in structure-guided drug design. Clemson University researchers led by professor Hugo Sanabria and working with researchers from Heinrich Heine University developed a hybrid fluorescence spectroscopic toolkit to study the essential reaction steps of enzymes. By combining single-molecule and ensemble multiparameter fluorescence detection, electron paramagnetic resonance spectroscopy, and Förster resonance energy transfer (FRET)-positioning and screening, as well as other biochemical and biophysical tools, they characterized three short-lived conformational states in the enzyme T4 lysozyme (T4L) over a nanosecond-millisecond timescale. T4L, an enzyme found in tears and mucus, is widely used to study protein structure and function because it’s such a stable enzyme. “We can track the lysozyme of the bacteriophage T4 as it processes its substrate at near atomistic level with unprecedented spatial and temporal resolution,” Sanabria said. The researchers placed two fluorescent markers on a set of molecules, creating a straight line to measure length at the molecular level. By using different marker locations, the team collected a set of distances that described the shape and form of the observed molecule. “We observe changes in the structure, and because our signal is time-dependent, we can also get an idea of how the molecule is moving over time,” Sanabria said. In essence, this process generated a collection of data points that were computationally processed, allowing the researchers to distinguish how the molecule looked and moved. The researchers engineered 34 different double-labeled samples for FRET experiments to measure distances across the enzyme to visualize its function. Each sphere represents one of the fluorescence markers with the corresponding engineered mutation site. Courtesy of Hugo Sanabria. The scientists found the structure of the T4L enzyme to be different than previously thought. “This molecule was considered a two-state molecule because of how it receives the substrate or cell wall of the target bacteria. However, we have identified a new functional state,” Sanabria said. Until now, scientists have determined the structures of proteins like T4L mainly through methods such as x-ray crystallography, nuclear magnetic resonance (NMR) spectroscopy, and cryo-electron microscopy. The team is helping to establish a database where its FRET-based structural biomolecular models can be stored and accessed by other scientists. The group is also working with others in the FRET community to establish recommendations for FRET microscopy. Clemson University biophysics associate professor Hugo Sanabria and an international team of researchers have demonstrated new optical imaging methods that may someday aid in structure-guided drug design. Courtesy of Ken Scar, Clemson University. Sanabria and his team hope to apply their imaging methodology to other biomolecules. “This optical method can be used to study protein folding and misfolding or any structural organization of biomolecules,” Sanabria said. “It can also be used for drug screening and development, which requires knowing what a biomolecule looks like in order for a drug to target it.” The spectroscopic toolkit developed by the researchers could accelerate development of dynamic structural biology by identifying transient conformational states that are abundant in biology and critical in enzymatic reactions. “This work is a milestone in structure determination using FRET to map short-lived functionally relevant enzyme states,” Claus A.M. Seidel, chair of the Institute for Molecular Physical Chemistry at Heinrich Heine University, said. The research was published in Nature Communications (www.doi.org/10.1038/s41467-020-14886-w).